Alamar Blue assay optimization to minimize drug interference and inter-assay viability.

Alamar Blue (AB) has become an increasingly popular reagent of choice for cell viability assays. We chose AB over other reagents such as MTT and Cell-Titer Glo due to its cost effectiveness and its ability to be a nondestructive assay. While analyzing the effect of osimertinib, an EGFR inhibitor on the non-small cell lung cancer cell line, PC-9, we noticed unexpected right-shifts of dose response curves as compared to the curve obtained by Cell Titer Glo assay. Here, we describe our modified AB assay method to avoid right shift right shift in dose response curve. Unlike some of the redox drugs that were reported to directly affected AB reading, osimertinib itself did not directly increase AB reading. Yet, the removal of the drug containing medium prior to AB addition eliminated falsely increased reading giving comparable dose response curve as the one determined by Cell Titer Glo assay. When a panel of 11 drugs were assessed, we found that this modified AB assay eliminated unexpected similar right shifts detected in other epidermal growth factor receptor (EGFR) inhibitors. We also found that plate-to-plate variability can be minimized by adding an appropriate concentration of rhodamine B solution to the assay plates to calibrate fluorimeter sensitivity. This calibration method also enables a continuous longitudinal assay to monitor cell growth or recovery from drug toxicity over time. Our new modified AB assay is expected to provide accurate in vitro measurement of EGFR targeted therapies.

Early outcomes of ultra-hypofractionated preoperative radiation therapy for soft tissue sarcoma followed by immediate surgical resection.

BACKGROUND: There is increasing interest in shorter courses of radiation therapy (RT) in the management of soft tissue sarcoma (STS). We report our institutional experience for patients undergoing ultra-hypofractionated preoperative RT followed by immediate resection. METHODS: An IRB approved review of patients treated with preoperative 5 fraction, once daily RT followed by immediate resection (within 7 days) for STS of the extremity or trunk was conducted. The primary endpoints are major wound complications and local control (LC). Secondary endpoints include grade ≥ 2 toxicity, metastasis free survival (MFS), and overall survival (OS). RESULTS: Twenty-two patients with a median age of 67 years (range 30-87) and median follow-up of 24.5 months (IQR 17.0-35.7) met eligibility criteria; 18/22 patients (81.8 %) had ≥ 1 year follow-up. Primary tumor location was lower extremity in 15 patients (68.2 %), upper extremity in 5 (22.7 %), and trunk in 2 (9.1 %). All patients received 30 Gy in 5 fractions. The median time to resection following RT was 1 day (range 0-5). The median time from biopsy to resection was 34 days (range 20-69). Local control was 100 %; in patients with localized disease, 2-year MFS and OS were 71.3 % and 76.9 %, respectively. Major wound complications occurred in 9 patients (40.9 %), with wound complications requiring reoperation occurring in 8 patients (36.4 %). Other acute and late grade ≥ 2 toxicities were seen in 0 and 4 patients (18.2 %), respectively. CONCLUSION: Ultra-hypofractionated preoperative RT followed by immediate resection permits expedited completion of oncologic therapy with early results demonstrating excellent local control and acceptable toxicity. Prospective data with long-term follow-up is needed.

Measuring competitive exclusion in non-small cell lung cancer.

In this study, we experimentally measure the frequency-dependent interactions between a gefitinib-resistant non-small cell lung cancer population and its sensitive ancestor via the evolutionary game assay. We show that cost of resistance is insufficient to accurately predict competitive exclusion and that frequency-dependent growth rate measurements are required. Using frequency-dependent growth rate data, we then show that gefitinib treatment results in competitive exclusion of the ancestor, while the absence of treatment results in a likely, but not guaranteed, exclusion of the resistant strain. Then, using simulations, we demonstrate that incorporating ecological growth effects can influence the predicted extinction time. In addition, we show that higher drug concentrations may not lead to the optimal reduction in tumor burden. Together, these results highlight the potential importance of frequency-dependent growth rate data for understanding competing populations, both in the laboratory and as we translate adaptive therapy regimens to the clinic.

UV decontamination of personal protective equipment with idle laboratory biosafety cabinets during the COVID-19 pandemic.

Personal protective equipment (PPE) is crucially important to the safety of both patients and medical personnel, particularly in the event of an infectious pandemic. As the incidence of Coronavirus Disease 2019 (COVID-19) increases exponentially in the United States and many parts of the world, healthcare provider demand for these necessities is currently outpacing supply. In the midst of the current pandemic, there has been a concerted effort to identify viable ways to conserve PPE, including decontamination after use. In this study, we outline a procedure by which PPE may be decontaminated using ultraviolet (UV) radiation in biosafety cabinets (BSCs), a common element of many academic, public health, and hospital laboratories. According to the literature, effective decontamination of N95 respirator masks or surgical masks requires UV-C doses of greater than 1 Jcm-2, which was achieved after 4.3 hours per side when placing the N95 at the bottom of the BSCs tested in this study. We then demonstrated complete inactivation of the human coronavirus NL63 on N95 mask material after 15 minutes of UV-C exposure at 61 cm (232 µWcm-2). Our results provide support to healthcare organizations looking for methods to extend their reserves of PPE.

Multidrug Cancer Therapy in Metastatic Castrate-Resistant Prostate Cancer: An Evolution-Based Strategy.

PURPOSE: Integration of evolutionary dynamics into systemic therapy for metastatic cancers can prolong tumor control compared with standard maximum tolerated dose (MTD) strategies. Prior investigations have focused on monotherapy, but many clinical cancer treatments combine two or more drugs. Optimizing the evolutionary dynamics in multidrug therapy is challenging because of the complex cellular interactions and the large parameter space of potential variations in drugs, doses, and treatment schedules. However, multidrug therapy also represents an opportunity to further improve outcomes using evolution-based strategies. EXPERIMENTAL DESIGN: We examine evolution-based strategies for two-drug therapy and identify an approach that divides the treatment drugs into primary and secondary roles. The primary drug has the greatest efficacy and/or lowest toxicity. The secondary drug is applied solely to reduce the resistant population to the primary drug. RESULTS: Simulations from the mathematical model demonstrate that the primary-secondary approach increases time to progression (TTP) compared with conventional strategies in which drugs are administered without regard to evolutionary dynamics. We apply our model to an ongoing adaptive therapy clinical trial of evolution-based administration of abiraterone to treat metastatic castrate-resistant prostate cancer. Model simulations, parameterized with data from individual patients who progressed, demonstrate that strategic application of docetaxel during abiraterone therapy would have significantly increased their TTP. CONCLUSIONS: Mathematical models can integrate evolutionary dynamics into multidrug cancer clinical trials. This has the potential to improve outcomes and to develop clinical trials in which these mathematical models are also used to estimate the mechanism(s) of treatment failure and explore alternative strategies to improve outcomes in future trials.